Because of the diversity of niches they inhabit, their versatility, and the threat their members pose to human health, Gram-positive bacteria are of paramount importance to multiple facets of microbiology. Nonetheless, there has been no systematic effort to elucidate gene functions and relationships on a genome-wide scale in any Gram-positive bacterium. Bacillus subtilis is the premier model organism for studies of Gram-positive bacteria because it offers powerful classical and molecular genetics, high-resolution cell biology, as well as a large and vibrant community of researchers. Evidence from other model organisms demonstrates that genome-wide approaches accelerate functional discovery. The opportunity for GO funding has catalyzed a broad-based community of researchers to pool their expertise to create a focused, coordinated, multidisciplinary effort to develop the genomic resources necessary to implement a comprehensive gene function effort. This initiative will serve as the blueprint for similar efforts in other model and pathogenic bacteria in the future. We will: 1. Construct genome-scale tools for global phenotypic analysis. These will include two bar-coded null mutant libraries, an ordered over-expression library, 300 promoter fusions to yfp and a C-terminal epitope- tagged library for all essential genes. 2. Develop and provide proof-of-principle experiments for key phenotyping methods. These will include high-throughput genetic interaction analysis and chemical genetic profiling;global cytological phenotyping;and transcriptional profiling. 3. Integrate and analyze the data across species. Each global phenotyping dataset will be analyzed in collaboration with bioinformaticists. This analysis will generate probabilistic functional interaction maps and transcriptional networks that will provide key information about gene functions and relationships. These will be used for comparative analysis among Gram-positive organisms and between Gram-positives and Gram- negatives. 4. Develop a B. subtilis database (subtilisHUB). We will build a web-based data resource to support ongoing functional annotation. This site will document the construction of all the genomic tools, house protocols and datasets, and provide infrastructure to enable Gene Ontology assignments, literature mining, and community discussion. 5. Engage the community of microbiologists in the genomic resources and approaches. A broad community of research specialists will launch genome-scale projects taking advantage of the tools, methodologies, and resources. PUBLIC HEALTH RELEVANCE: This proposal is aimed at elucidating the function of all the genes in Bacillus subtilis, the major model organism for an important class of bacteria, Gram-positive bacteria. The project will generate genome-scale tools;pioneer the development of global phenotypic analysis for bacteria;and establish a web resource that will ensure continuity beyond the funding term. The concerted, community-based effort we propose will have far-reaching impact on research in bacteria in general, and on many Gram-positive pathogens.